Link to this post | posted 06 Aug, 2020 19:57

c.sunnen

Hi! In preparation for running some investigations beyond annotation this coming semester, we've been working though the suggestions in the 2013 "Exploring bacteriphage biology" document found on phagesdb, and we've been working through softberry to find terminators. We'd like to write up a "protocol" for our undergrads, but we're having a hard time finding info on how to interpret the data. Who else has used this program, and can you point us to pre-written protocols/resources? Most papers we found don't explain how they used it at all. Thanks! Nikki

Link to this post | posted 03 Jun, 2020 19:07

c.sunnen

Hi! While it makes no sense whatsoever, I'm getting some good hits in HHPred to "Caudovirus prohead serine protease" (98% probability, ~95% coverage; see attachment) for GC phage Rasputia gp130 (in phagesdb and Phamerator gp130. That's the same as "capsid maturation protease," no? Note that this is an orpham, and we've already got a capsid maturation protease (where it should be!) I'd like to call this simply "peptidase" for now, but I'd love a second opinion on this. It's such a good match in HHPred! 62Kb

Link to this post | posted 23 Mar, 2020 20:08
c.sunnen	Thanks, Debbie! I totally agree that it's not worth calling at this point, but boy is it an interesting case! As always, I appreciate the experts weighing in. Hope things are well!

Link to this post | posted 23 Mar, 2020 19:46

c.sunnen

Rasputia (GC cluster) appears to have 4 tRNAs in a cluster (see my attachment with screenshots from DNAMaster and tRNAScanSE). The one in question is what DNAMaster is calling feature 150. It appears to have an intron? It is way too long, and online Aragorn does not call it. BUT, tRNAScanSE DOES, and gives it a good infernal score (43. and a mostly cononical structure. However, it doesn't appear to have an anticodon. My hunch is that it's not real, but if we delete it, we'd be leaving a big hole. I know that we generally don't call it unless it's less than 90bp, but I'm really curious if this could be at all real. Unless I hear otherwise, we won't call it. But I'd love to know more if anyone can offer some insight. Thanks! Nikki 159Kb

Link to this post | posted 14 Nov, 2019 03:22

c.sunnen

Hi! My first suggestion is to digest longer; we typically do ours for at least an hour at 37deg or overnight at room temp. Good news is your DNA looks nice! Some smearing, but mostly intact. If you're uncertain if your enzyme is any good, you can try digesting some lambda DNA (provided you have some on hand). I hope this is helpful! Good luck! Nikki P.S. If you're using the protocol I've posted above, there have been a few edits, though it should still work in its current form.

Link to this post | posted 12 Jul, 2019 15:29

c.sunnen

I had something similar in Mashley (EG). Genes 42 and 43 were orphams, the result of a frameshift that split a gene in approximately half. Interestingly, they split a "membrane protein" into two "membrane protein" proteins! Both orphams retained several TM domains! So they both got called that way. In your case, I think calling them both NKF genes is probably appropriate (unless there is a function you can attribute to either/both combined). Without bench data to suggest otherwise, as long as it's got coding potential, it's probably safer to assume it does get made, even if it's non-functional.

Link to this post | posted 11 Jul, 2019 21:12

c.sunnen

Hi! I am QC-ing EE phage Vanisius, and have some good HHPred hits to "tail fiber" for gp 15 (see screenshots in first slide of attached PPT). Most are calling this pham simply a minor tail protein, but while it's in the right place, it's only ~550bp. What's the expected size for a tail fiber? Is this best called NKF, minor tail, or tail fiber? In a related question, gp 9 has some good HHPred evidence for a minor tail protein of some kind (see the 2nd slide in attachment), but is less than 400bp long. It is immediately downstream of the major tail protein and immediately upstream of the tail assembly chaperones. I want to confirm that this is best left as NKF? Thanks! 156Kb

Link to this post | posted 30 May, 2019 19:28
c.sunnen	Matt, Did you use our protocol? Your gel looks similar to ours. We also had some issues with sequencing - turns out we had some bacterial DNA contamination - but we assumed that was user error (asking the TAs to add the nuclease instead of doing it ourselves). We're actually going to test this over the summer. Otherwise, our DNA seemed pretty clean. Nikki

Link to this post | posted 29 May, 2019 20:19
c.sunnen	On the large gel (previous post, 1.2% agarose), after the ladder, each sample set is loaded as: water uncut, NspI, SacII. On the small gel (attached, 2% agarose), the sets are loaded as: buffer uncut, HaeIII. So large gel lanes 1-3 and small gel lanes 1-2 are the same phage. 264Kb

Link to this post | posted 29 May, 2019 20:18
c.sunnen	On the large gel (attached, 1.2% agarose), after the ladder, each sample set is loaded as: water uncut, NspI, SacII. On the small gel (next post, 2% agarose), the sets are loaded as: buffer uncut, HaeIII. So large gel lanes 1-3 and small gel lanes 1-2 are the same phage. 264Kb